对与缺失铁钼辅基的固氮酶钼铁蛋白重组的原子簇的结构要求

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）钼铁蛋白与邻菲口罗啉和Ｏ２ 保温并经凝胶柱层析后,成为部分缺失Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ 的失活蛋白。由３ 个－ ＯＣＨ３－ 连结于Ｍｏ原子间的２ 个１Ｍｏ∶３Ｆｅ∶４Ｓ结构单位组成的原子簇与４Ｆｅ∶４Ｓ原子簇的混合液,以及由ＫＭｎＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液均可使这种失活蛋白明显恢复Ｃ２Ｈ２ 还原活性,但都不能使可被ＦｅＭｏｃｏ 激活的ｎｉｆＥ和ｎｉｆＨ 基因缺失突变种及ＵＷ ４５的Ａｐｏ－ＭｏＦｅ 蛋白得到激活。表明,不能合成ＦｅＭｏｃｏ 的固氮菌突变种蛋白只能与具有ＦｅＭｏｃｏ相似或基本相似结构的原子簇进行体外重组,而部分缺失金属原子簇的钼铁蛋白能与具有一定结构和组成的原子簇进行体外重组,变为具有催化氮还原能力的各种固氮酶组分蛋白。     

锰对部分缺失金属原子簇的固氮酶钼铁蛋白的重组作用

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ 的失活蛋白,经与由ＫＭｎＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,重组蛋白的吸收光谱和对Ｃ２Ｈ２、Ｈ＋ 和Ｎ２ 的还原活性都恢复至与还原钼铁蛋白相似的状态,而它的α－螺旋度和在３８０—５５０ ｎｍ 、６２０—６７０ ｎｍ 的ＣＤ谱虽有明显的恢复,但仍与还原钼铁蛋白有所不同。表明：（１）重组蛋白液既含有在缺失金属原子簇的ＭｏＦｅ蛋白与含Ｍｎ 重组液重组过程中可能组装的ＭｎＦｅ 蛋白,又含有在邻菲口罗啉和Ｏ２ 处理后金属原子簇仍旧完整的ＭｏＦｅ蛋白;（２）ＭｎＦｅ蛋白和ＭｏＦｅ蛋白在固氮能力上可能是相似的,而在结构上却可能略有差异     

异源nifA^C对根癌土壤杆菌nif基因表达的调节作用

用三亲水交配方法分别将载有褐球固氮菌（Ａｚｏｔｏｂａｃｔｅｒｃｈｒｏｏｃｏｃｃｕｍ）呈组成型表达的ｎｉｆＡＣ的质粒ｐＣＫ５和肺炎克氏杆菌（Ｋｌｅｂｓｉｅｌｌａｐｎｅｕｍｏｎｉａｅ）含有ｎｉｆＡ＾Ｃ和ｎｉｆＡ－ｎｔｒＣ基因的质粒ｐＣＫ３,ｐＳＺ３６和ｐＳＺ２３－ＣＡ导入根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｎｅｆａｃｉｅｎｓ）Ｃ５８／ｐＧＶ３８５０所得转移接合子的生长速率和野生型相似。在１０ｍ     

含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失ＦｅＭｏｃｏ 和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬（ＣｒＦｅ）蛋白     

钒铁蛋白的体外组装研究

棕色固氮菌固氮酶ＭｏＦｅ蛋白经邻菲罗啉在厌氧和有氧条件下处理后,成为部分缺失ＦｅＭｏｃｏ和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白,用由钒酸钠,高柠檬酸铁、Ｎａ２Ｓ和二硫硫苏糖醇组成的溶液保温后,可使这失活蛋白变为重组蛋白。重组蛋白的ＣＤ谱除５５０－６５０ｎｍ与ＭｏＦｅ蛋白存在差异外,也能与其吸收光谱和对质子还原活性一样得到基本恢复,但对Ｃ２Ｈ２的还原活性却未得到明显恢复。表明,这种重组蛋白与ＭｏＦｅ蛋白有所     

锰对部分缺失金属原子族的固氮酶钼铁蛋白的重组作用

棕色固氮菌固氮酶钼铁蛋白经邻菲罗啉的Ｏ２处理后,变为部分缺失Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ的失活蛋白、经与由ＫＭｎＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,重组蛋白的吸收光谱和对Ｃ２Ｈ２、Ｈ＋和Ｎ２的还原活性都恢复至与还原钼铁蛋白相似的状态,而它的α－螺旋度和在３８０－５５０ｎｍ,６２０－６７０ｎｍ的ＣＤ谱虽有明显的恢复,但仍与还原钼铁蛋白有所不同。表明：（１）重组蛋白液既含有在     

锰,铬和钼重组液及其与部分缺金属原子簇钼铁蛋白重组的比较研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒｖｉｎｅｌａｎｄｉ）钼铁蛋白经邻菲口罗啉和Ｏ２处理后,缺失一部分ＦｅＭｏｃｏ和Ｐｃｌｕｓｔｅｒ,并失去大部分活性。高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇（ＤＴＴ）分别与Ｋ２ＣｒＯ４、ＫＭｎＯ４和Ｎａ２ＭｏＯ４按一定比例混合的具有不同颜色的重组液,均可显著激活失活的钼铁蛋白;然而,除ＤＴＴ可略提高失活蛋白的活性外,其它化合物或缺一、二种化合物的混合液均无激活能力。含９９Ｍｏ重组液与失活蛋白重组后的微分扰动角关联测定显示,该蛋白中的４１％的９９Ｍｏ的状态与克氏肺炎杆菌９９ＭｏＦｅ蛋白中的９９Ｍｏ相似,表明这种激活主要是以金属原子簇含量恢复为基础的。由此可推论出,含Ｍｎ或Ｃｒ的重组液,通过与失活钼铁蛋白重组得到含Ｍｎ或Ｃｒ的固氮酶是可能的。     

棕色固氮菌钼铁蛋白的金属原子簇与其构象的关系

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白的紫外ＣＤ谱在２０５—２３５ ｎｍ 出现由峰位分别为２０８ 和２２２ ｎｍ 的双负峰组成的负槽;经邻菲口罗啉在厌氧或有氧环境中处理后,在２２２ ｎｍ 的负峰随该蛋白中Ｐ－ｃｌｕｓｔｅｒ 和ＦｅＭｏｃｏ 含量的降低而减少。在分别由Ｎａ２ＭｏＯ４、Ｎａ２Ｓ、二硫苏糖醇和高柠檬酸铁或柠檬酸铁组成的重组液重组后,上述两种处理蛋白在２２２ ｎｍ 的负峰又随其Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ含量的恢复而恢复。表明,钼铁蛋白的金属原子簇与蛋白质构象间存在明显的相关性     

尿素对钼铁蛋白的活性和金属原子簇稳定性的影响

当尿素浓度高于０．５ ｍ ｏＬ／Ｌ时,棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白的乙炔还原活性呈指数下降;而经厌氧缓冲系统稀释并保温后,又可得到明显恢复。尿素对邻菲口罗啉从还原的或部分缺失Ｐ－ｃｌｕｓｔｅｒ 的ＭｏＦｅ 蛋白中螯合金属原子簇的Ｆｅ 原子均有较大的促进作用。还原ＭｏＦｅ 蛋白在尿素梯度凝胶电泳中的迁移率：在０～１．５ ｍ ｏｌ／Ｌ内,无明显变化;在１．５～５．０ ｍ ｏｌ／Ｌ内,线性变小;在５．０～８．０ ｍ ｏｌ／Ｌ内,则呈平缓状态。结果表明：（１）尿素对不同状态的ＭｏＦｅ 蛋白金属原子簇的影响程度不尽相同,而对蛋白质的变构作用是尿素影响ＭｏＦｅ 蛋白的活性和金属原子簇稳定性的主要原因;（２）ＭｏＦｅ 蛋白的构象与金属原子簇密切相关;（３）ＭｏＦｅ 蛋白在变性过程中,活性下降可能先于分子整体构象的变化     

含铬重组液激活部分缺陷失金属原子族的钼铁蛋白的研究

棕色固氮菌固氮酶相铁蛋白经邻菲罗啉和Ｏ２处理,变为部分缺失ＦｅＭｏｃｏ和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显恢复,然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也话存在功能与钼铁蛋白相似,而结构则有所差异的含铬蛋白。     

Characteristics and Crystallization of Nitrogenase MoFe Protein from a nif Z Deleted Strain of Azotobacter vinelandii

△nifZ MoFe protein purified from a nifZ deleted strain of Azotobacter vinelandii (DJ194) was shown to be pure by SDS-Polyacrylamide gel electrophoresis. The protein contained 1.5 Mo atoms and 15.9 Fe atoms per molecule, the ratio of Fe to Mo was lower than that of the MoFe protein purified from the wild type strain of A. vinelandii; and Call2, H+ -reduction activity and their ratio (C2H4/H2 (Ar)) were 16.6%, 21.7% and 77.2% of those of the wild type MoFe protein, respectively. Under a somewhat different condition from that for the crystallization of the wild type MoFe protein dark brown rhombohedron crystals of △nifZ MoFe protein were obtained. It indicated that the deletion of the △nif Z resulted in the decrease of number or change in the structure of P-cluster in the mutant MoFe protein, which caused the significant structured and function of change of the protein.     

Purification and Properties of Nitrogenase MoFe Protein from a nif Z Deletion Strain of Azotobacter vinelandii

The MoFe protein of the nif Z deletion strain (△nif Z MoFe protein) of Azotobac ter vinelandii designated DJ 194 was purified and some properties were studied. The cell free extract of DJ 194 was more sensitive to O2 and heat than the wild-type extract. The specific activity of the purified DJ 194 protein was 283 nmol C2H2 reduced/(min · mg protein), which was much lower than that of purified wild-type A. vinelandii MoFe protein. The △nif Z MoFe protein exhibited a visible similar absorption spectra as the wild type MoFe protein, yet showed significant difference in CD and MCD spectra at the region about 450 mm com paring with the spectral property of the wild-type MoFe protein. This seems to indicate that the P-cluster of the △nif Z MoFe protein was modified, which might be the cause of the low activity of the DJ 194 MoFe protein.     

Purification and Characteristics of Mn-containing Nitrogenase Component

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but undergo phenotypic reversal to Nif+ under Mo deficiency, was able to grow in Mo- and NH3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C2H2- and H+-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰprotein.     

Relationship Between nifZ and the Synthesis of P cluster in Nitrogenase MoFe Protein of Azotobacter vinelandii

In comparison with OP MoFe protein from wild type strain Azotobacter vinelandii Lipmann, the C2H2-reduction activity and atom ratio of Fe to Mo of △nifZ MoFe protein from a nifZ deletion strain of A. vinelandii were remarkably decreased. FeMoco, which were extracted from these two proteins under the same condition, were almost similar to each other in activity and metal composition, and the circular dichroism (CD) spectra of these proteins were significantly different from each other. In the visible region except 540 750 nm, the △ε at 380 - 540 nm of △nifZ MoFe protein decreased and had a peculiar sharp negative peak around 430 nm; and in the ultraviolet region, the peaks at 208 nm and 222 nm were higher than those of OP MoFe protein. △nifZ MoFe protein could be crystallized in a suitable concentration of PEG 8000 and MgCl2, the size of crystals and amount of precipitation seemed to be related to the above-mentioned negative peaks. The results showed that △nifZ of Azotobacter vinelanclii might be related to the synthesis of P-cluster, rather than to that of FeMoco, which resulted in its conformation, stability and process of crystallization.     

Purification and Characterization of Cr-containing Nitrogenase Component I

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but can undergo phenotypic reversal to Nif+ under Mo deficient conditions, was able to grow in Cr containing but Mo and NH3 deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW3 grown on the Cr containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C2H2 and H+ reduction activity of MoFe protein from the wild type strain of Azotobacter vinelandii Lipmann. The Cr containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two differentsubunits (α2β2). The preliminary results indicated that the Cr containing protein might be a nitrogenase component Ⅰ protein.     

缺失nifE的棕色固氮菌突变种MoFe蛋白的纯化及体外激活

经DEAE纤维素、Sephacryl S-300和Q-Sepharose柱层析分离纯化,从缺失nifE的棕色固氮菌(Azotobactervinelandii Lipmann)突变种(DJ35)的无细胞粗提物中得到△nifE MoFe蛋白(△nifE Av1).SDS凝胶电泳分析表明,△nifE Av1的亚单位种类和分子量分别与棕色固氮菌野生型(OP)MoFe蛋白(Av1)的α和β亚单位相似.当与固氮酶Fe蛋白(Av2)活性互补时,△nifE Av1不具有还原质子的能力,但从OP Av1中抽提的FeMoco却可使其激活.经过量的邻菲啰啉(o-phen)厌氧处理并经Sephadex G-25柱层析分离后,便得到△nifE Av1 .在同时存在Av2和MgATP发生系统的条件下,△nifE Av1 ,而不是△nifE Av1,可为由KMnO4、高柠檬酸铁、Na2S、Na2S2O4和二硫苏糖醇组成的含Mn重组液(RS-Mn)显著激活.但在缺少MgATP或Av2的条件下,RS-Mn则不能激活△nifE Av1 .这就表明,RS-Mn对△nifE Av1 的激活需要o-phen的预先处理及同时存在Av2和MgATP的这二个条件.     

Purification and properties of the nitrogenase of Azospirillum amazonense.

The nitrogenase of the free-living, microaerobic, N2-fixing bacterium Azospirillum amazonense (strain Y1) was purified by chromatography on DEAE-52 cellulose, by heat treatment, and by preparative polyacrylamide gel electrophoresis. The specific nitrogenase activities were 2,400 nmol of C2H4 formed per min per mg of protein for dinitrogenase (MoFe protein) and 1,800 nmol of C2H4 formed per min per mg of protein for dinitrogenase reductase (Fe protein). The MoFe protein was composed of a minimum of 1,852 amino acid residues, had an isoelectric point of 5.2, and contained 2 atoms of Mo, 24 atoms of Fe, and 28 atoms of acid-labile sulfide per molecule. The Fe protein had 624 amino acid residues and an isoelectric point of 4.6 and contained four atoms of Fe and six atoms of acid-labile sulfide per molecule. The purified MoFe protein showed two subunits with molecular weights of 55,000 and 50,000. The purified Fe protein revealed two polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weights of 35,000 and 31,000. The two Fe protein polypeptides were demonstrated with immunological techniques in the purified, highly active enzyme as well as in extracts. Also, Azotobacter vinelandii Fe protein showed two closely migrating polypeptides that migrated differently from the Fe protein polypeptides of Azospirillum brasilense or Rhodospirillum rubrum. The nitrogenase activity of Azospirillum amazonense Y1 was independent of Mn2+, and the addition of activating enzyme had no effect. No activating enzyme could be found in Azospirillum amazonense. Obviously, the nitrogenase system of Azospirillum amazonense Y1 is different from that of Azospirillum brasilense Sp7 and resembles the Azotobacter system.     

Identification of an Fe protein residue (Glu146) of Azotobacter vinelandii nitrogenase that is specifically involved in FeMo cofactor insertion

The Fe protein of nitrogenase has three separate functions. Much is known about the regions of the protein that are critical to its function as an electron donor to the MoFe protein, but almost nothing is known about the regions of the protein that are critical to its functions in either FeMo cofactor biosynthesis or FeMo cofactor insertion. Using computer modeling and information obtained from Fe protein mutants that were made decades ago by chemical mutagenesis, we targeted a surface residue Glu(146) as potentially being involved in FeMo cofactor biosynthesis and/or insertion. The Azotobacter vinelandii strain expressing an E146D Fe protein variant grows at approximately 50% of the wild type rate. The purified E146D Fe protein is fully functional as an electron donor to the MoFe protein, but the MoFe protein synthesized by that strain is partially ( approximately 50%) FeMo cofactor-deficient. The E146D Fe protein is fully functional in an in vitro FeMo cofactor biosynthesis assay, and the strain expressing this protein accumulates "free" FeMo cofactor. Assays that compared the ability of wild type and E146D Fe proteins to participate in FeMo cofactor insertion demonstrate, however, that the mutant is severely altered in this last reaction. This is the first known mutation that only influences the insertion reaction.     

The Effect of Urea on the Activity and Stability of Metallocluster of the MoFe Protein of Nitrogenase from Azotobacter vinelandii

When the MoFe protein from Azotobacter vinelandii was treated with more than 0.5 mol/L of urea anaerobically, the C2H2-reduction activity of the treated protein was exponentially decreased. However, the activity could be significantly restored after the treated protein was diluted with the buffer system and followed by incubation at 15 ℃. The urea had remarkable enhancement on chelation of Fe atoms from the reducted MoFe protein and the P-cluster-deficient MoFe protein by O-phenanthroline (O-phen), respectively. The migration of the reducted MoFe protein on the urea-gradient gel electrophoresis did not significantly change from 0 to 1.5 mol/L urea , linearly became smaller from 1.5 to 5.0 mol/L urea, and reached a stable state from 5.0 to 8.0 mol/L urea. The results indicated that: (1) The effect of urea on the activity and the stability of metallocluster of the MoFe protoin was mainly attributed to the conformational change of the protein in urea, moveover, the effect of urea on the metallocluster might not be all the same if the states of MoFe protein were dif ferent; (2) There was a close relationship between the conformation and the metalloclusters of MoFe protein; (3) In the course of the denaturation, the decrease in the activity of MoFe protein probably happened prior to the conformational change of the whole molecule.     

Azotobacter vinelandii nitrogenases containing altered MoFe proteins with substitutions in the FeMo-cofactor environment: effects on the catalyzed reduction of acetylene and ethylene

Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C(2)H(2), C(2)H(4), CO, and H(2). The altered MoFe proteins used were the alpha-195(Asn) or alpha-195(Gln) MoFe proteins, which have either asparagine or glutamine substituting for alpha-histidine-195, and the alpha-191(Lys) MoFe protein, which has lysine substituting for alpha-glutamine-191. On the basis of K(m) determinations, C(2)H(2) was a particularly poor substrate for the nitrogenase containing the alpha-191(Lys) MoFe protein. Using C(2)D(2), a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C(2)D(2)H(2), and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the alpha-195(Asn) or the alpha-191(Lys) MoFe proteins, which also exhibited the highest rate of ethane production from C(2)H(2). These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C(2)H(4) was not a substrate of the nitrogenase with the alpha-191(Lys) MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the alpha-195(Gln) MoFe protein, both of which had a low V(max) and high K(m) (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the alpha-195(Asn) MoFe protein, which exhibited a K(m) of 48 kPa and a specific activity for C(2)H(6) formation from C(2)H(4) 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the alpha-195(Asn) MoFe protein produced cis-C(2)D(2)H(2) when turned over under trans-C(2)D(2)H(2). These results suggest that the C(2)H(4)-reduction site is affected by substitution at residue alpha-195, although whether the effect is related to the substrate-reduction site directly or is mediated through disturbance of the delivery of electrons/protons is unclear. Ethylene inhibited total electron flux, without uncoupling MgATP hydrolysis from electron transfer, to a similar extent for all four A. vinelandii nitrogenases. This observation indicates that this C(2)H(4) flux-inhibition site is remote from the C(2)H(4)-reduction site. Added CO eliminated C(2)H(4) reduction but did not fully relieve its electron-flux inhibition with all four A. vinelandii nitrogenases, supporting the suggestion that electron-flux inhibition by C(2)H(4) is not directly connected to C(2)H(4) reduction. Thus, C(2)H(4) has two binding sites, and the presence of CO affects only the site at which it binds as a substrate. When C(2)H(2) was added, it also eliminated C(2)H(6) production from C(2)H(4) and also did not relieve electron-flux inhibition fully. Thus, C(2)H(2) and C(2)H(4) are likely reduced at the same site on the MoFe protein. Two schemes are presented to integrate the results of the interactions of C(2)H(2) and C(2)H(4) with the MoFe proteins.